1. Field of the Invention
The present invention relates to chemical dyes which can be used as fluorescent markers, and more particularly relates to monomethine cyanine-based compounds which have been sterically rigidized by the inclusion of a bridging boron atom between the compound's heterocyclic groups and which may be produced or chemically modified to include reactive or other groups to allow the compounds to covalently or noncovalently associate with a material to thereby impart fluorescent properties to the material.
2. Description of the Invention Background
Fluorescent dyes are generally known for imparting fluorescence to biological and nonbiological materials and have been used to detect various biological or other materials by procedures such as fluorescence microscopy, fluorescence immunoassay techniques, and flow cytometry. The primary advantages of fluorescent dyes compared with dyes detectable only through light absorption include (i) the emission of light by fluorescent dyes at a wavelength different from their excitation wavelength; (ii) the greatly enhanced detectability of fluorescence emission compared to light absorption; and (iii) the generally minimal level of background fluorescence in most biological materials.
A common method for labeling biological and nonbiological materials with fluorescent dyes is to create a fluorescent complex through covalent bonding between groups on the dye molecules and compatible groups on the material. In this way, material such as, for example, cells tissues, amino acids, proteins, antibodies, enzymes, drugs, hormones, nucleotides, nucleic acids, polysaccharide, and lipids may be chemically labeled and quantified or may be used as fluorescent probes that bind specifically to target materials (genetic sequences, haptens, antibodies, analytes, etc.) that are to be detected by fluorescence methods. Polymer particles, cells, and other materials labeled with fluorescent dyes can also be used as fluorescent standards in flow cytometers, imaging microscopes, and other fluorescence-based detection equipment. Minute fluorescent polymer particles can also be used as labels in immunofluorescence tests, toxicology testing, and analysis of genetic sequences. Because of their utility in research and medicine, a large market has developed for fluorescent reagents including prepared protein, antibody, and nucleic acid, and other probes pre-labeled with detectable fluorescent or fluorescent dye compounds.
Because most dye molecules are either nonfluorescent or only weakly fluorescent, available fluorescent dye markers have been derived from a relatively limited number of fluorescent aromatic structures. New fluorophores with optimal properties are rarely developed. Two common classes of fluorescent dyes are those derived from the fluorescein and rhodamine chromophores. Fluoresceins fluoresce green light whereas rhodamines fluoresce in the green-orange and red regions of the spectrum. The rhodamines are difficult labeling reagents to use, are not particularly fluorescent when bound to proteins, and often cause the precipitation of the labeled protein when the dye-to-protein ratio is greater than 2:1. One particular fluorescein dye, fluorescein isothiocyanate ("FITC"), and its conjugates, enjoy wide acceptance primarily because they have a relatively high extinction coefficient and have a high quantum yield. (Quantum yield is generally related to a molecule's rigidity or planarity and indicates the molecule's propensity to fluoresce, i.e., give off energy as light, rather than give off heat when energy is provided to the molecule.) However, fluorescein dyes have a number of disadvantages, including their strong tendency to photobleach when illuminated by a strong excitation source such as the lamps used in fluorescence microscopes. When a fluorescent compound photobleaches, a large percentage of the compound's fluorescence may be lost within seconds of illumination, resulting in a rapidly diminishing image. Also, when performing fluorescence assays, the loss of image through time by photobleaching makes quantifying results much more difficult and will ultimately result in a decreased ability to detect the analyte. Reagents, such as propyl gallate and p-phenylene-diamine, may retard but do not entirely eliminate photobleaching. The fluoresceins also have a pH-sensitive absorption spectrum and fluorescence yield decreases below pH 8 and the fluoresceins do not fluoresce at low pH.
Multiple fluorophores of different colors are used simultaneously in multi-parameter analyses for detecting and correlating different fluorescently-labeled materials in such procedures as flow cytometry, microscopy, chromatography and various other detection systems. In multi-parameter analyses, a number of fluorescent compounds having a binding affinity for different targets and having different maximum emission wavelengths are used to detect and quantify the sample's various targets. To reduce the overlap of fluorescence signals in multi-parameter analyses that are emitted from the target materials labeled with different fluorescent compounds, it is desirable to use fluorescent compounds with narrow absorption and emission bands.
One class of blue-fluorescing dyes, the coumarins, suffer from a number of disadvantages. For example, the coumarin-based fluorophore 7-amino-4-methylcoumarin acetate has broad absorption and emission peaks. Also, this compound has a relatively low extinction coefficient of approximately 17,000 l/mol-cm (Handbook, of Fluorescent Probes and Research Chemicals, Molecular Probes Inc., Eugene, Oreg.), thereby providing a fluorescence capacity (equal to the mathematical product of the extinction coefficient and the quantum yield) of approximately one-quarter that of the present inventions' compounds. Similarly, a fluorescent labeling dye available from Molecular Probes, Inc., under the trade name Cascade Blue (having the structure shown below where R is a reactive group) has an extinction coefficient of only 29,000 l/mol-cm. ##STR2##
The cyanine compounds are now recognized as fluorescent labeling dyes. Cyanines generally include two heterocyclic groups connected by a chain of conjugated double bonds with an odd number of carbon atoms and have been used as spectral sensitizers for photographic film. Cyanine compounds are utilized as spectral sensitizers in, for example, U.S. Pat. Nos. 4,337,063 (Miraha et al.) and 4,404,289 (Masuda et al.), 4,405,711 (Masuda et al.), and British Patent No. 1,529,202 (Exekial et al.). Fluorescence is not necessary for the photographic applications in those patents and fluorescent properties are not mentioned in those patents. The utility of cyanine compounds as fluorescent dyes was discovered only recently.
Cyanine compounds known to be useful fluorescent dyes include the unrigidized, arylsulfonated cyanine compounds of U.S. Pat. No. 5,268,486 to Waggoner et al, having the following general structure: ##STR3##
wherein, the dotted lines represent one to three rings having five to six atoms in each ring. R.sub.3, R.sub.4, R.sub.8 and R.sub.9 groups are attached to the rings. At least one of the R.sub.8 and R.sub.9 groups is a sulfonic acid or sulfonate group and at least one of the R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.7 groups is a moiety that will react with amino, hydroxy, phosphoryl, or sulfhydryl groups. The Waggoner et al. patent relates to trimethine, pentamethine, etc., cyanines (i.e., cyanines with more than a single double bond in the conjugated chain) and which fluoresce in the green, orange, red and near-infrared regions of the spectrum. Cyanines of this type have not been considered useful covalent labels.
One class of the cyanines includes a single atom bridging the heterocycles. These compounds are referred to herein as "rigidized" cyanines because the bridging atom restricts movement of the heterocycles about the conjugated carbon atom chain. Certain rigidized cyanines have been developed for photographic sensitization. See U.S. Pat. Nos. 2,541,400 to Brooker et al. and 3,148,187 to Heseltine. These cyanines cannot be used as fluorescent labeling dyes.
A group of rigidized cyanines having a single conjugated carbon atom linking the heterocyles ("rigidized monomethine cyanines") are those which are rigidized by incorporation of a boron molecule. Such compounds include pyrrole-based fluorophores derived from the bispyrromethene borondifluoride (2,2'-pyrromethene-1,1'-borondifluoride) complex having the following structure, where R represents various possible substituents: ##STR4##
Particular derivatives of the bispyrromethene borondifluoride structure include 3,3',5,5'-tetramethyl-2,2'-pyrromethene-1,1'-boron-difluoride, sold under the trademark BODIPY by Molecular Probes, Inc., Eugene, Oreg. BODIPY analogs are disclosed in U.S. Pat. No. 4,774,339 to Haugland and Kang, as well as in "Handbook of Fluorescent Probes and Research Chemicals" compiled by Haugland and published by Molecular Probes, Inc. The application of pyrrole-based boron complexes as laser dyes and in photodynamic therapy is described in U.S. Pat. No. 4,916,711 to Boyer and Morgan. The lowest wavelength BODIPY molecule has been shown to have a maximum absorptive wavelength of 500 nm and a maximum emissive wavelength of 508 nm in aqueous media, i.e., in the "green" region of the spectrum. The synthetic scheme for producing the above pyrrole-based boron complexes is provided in Shah et al., "Pyrromethene-BF.sub.2 Complexes as Laser Dyes", Heteroatom Chemistry, Vol. 1, Number 5 (1990) pages 389-399. Synthetic methods of the various boron complexes related to the BODIPY complexes are described in U.S. Pat. Nos. 4,774,339, 5,187,228, 5,248,782 and 5,274,113, all of Haugland et al.
The accepted general synthetic method for all bispyromethene boron complexes is shown below, where R.sub.1 through R.sub.7 are hydrogen or other groups or chromophores. ##STR5##
U.S. Pat. No. 4,774,339 describes pyrrole-based dyes which absorb and emit comparable to fluorescein, i.e., with maximum absorbance at approximately 490 nm and maximum emission at approximately 500 nm, and which include no additional chromophores attached to the basic structure shown in the general synthetic method above. U.S. Pat. No. 5,187,288 describes the synthesis of longer wavelength pyrrole-based boron complexes having additional chromophores at the R.sub.1 and/or R.sub.6 sites. Typical chromophores include phenylbutadienyl and phenylethenyl groups. U.S. Pat. No. 5,248,782 provides a synthetic method for longer wavelength pyrrole-based boron complexes dyes wherein heterocyclic bases are attached at R.sub.1 and R.sub.6. Typical examples of the heterocyclic bases include ##STR6##
As disclosed in Kang and Haugland, "Spectral Properties of 4-Sulfonato-3,3'5,5'-Tetramethyl-2,2'-Pyrromethene-1,1'-Borondifluoride Complex (Bodipy), Its Sodium Salt, and Protein Derivatives", SPIE Vol. 1063 New Technologies in Cytometry (1989), pages 68-73, the BODIPY compound and its sulfonated derivative have the spectral properties provided in Table 1 and the sulfonated BODIPY derivative has the absorption and emission spectrum depicted in FIG. 1. The notation "RI" in Table 1 indicates that the values provided are relative intensities with 1.00 being the fluorescence intensity of the particular dye in methanol at the dye's maximum emissive wavelength.
TABLE 1 Spectral Data For BODIPY And Its Sulfonate Derivative Max. Max. absorption Extinction emission wavelength coefficient wavelength Solvent (nm) (cm.sup.-1 mM.sup.-1) (nm) RI Bodipy- sulfonate: Water 494 39.7 509 0.67 Methanol 500 57.7 515 1.00 Acetonitrile 504 58.9 514 0.69 Dioxane 506 52.7 518 0.64 Bodipy: Water 500 -- 508 0.48 Methanol 501 85.3 509 1.00 Acetonitrile 501 86.7 508 0.87 Dioxane 505 80.0 512 0.63
A pyridine-based monomethine boron complex bis-benzomethene borondifluoride having the following structure ##STR7##
was reported by Bashing, Schafer, and Steyer, Applied Physics, volume 3, p. 81 (1974). The synthetic scheme for producing the pyridine based compound is complex and was reported by Douglas (Journal of Heterocyclic Chemistry, Vol. 10, p. 255 (1973)).
Quinoline-based monomethine boron complexes having the following structures were reported by Scheibe and Daltrozzo, Advances in Heterocyclic Chemistry, volume 6, p. 153 (Academic Press 1965). However, their synthesis was never reported. ##STR8##
Although these quinoline-based compounds have been evaluated for use as laser dyes, it is unknown whether they are useful as fluorescent dye markers. Based on their structure, it is believed that the above quinoline-based molecules would have a maximum emissive wavelength significantly greater than 500 nm.
There is an absence of bright, soluble fluorescent dye compounds that fluoresce in the shorter wavelength (300-500 nm) region of the spectrum, have desirable spectral properties and can be used to impart fluorescence to a large number of materials. One object of the present invention is to provide a class of bright, highly fluorescent, strongly light-absorbing dyes that will emit in the spectrum's blue region, and that can be useful in a variety of biological and nonbiological applications. It is also an object of the invention to provide soluble fluorescent markers which have maximum absorptive and emissive wavelengths in the blue spectral region below 500 nm.
It is also an object of the present invention to provide fluorescent markers which can covalently or noncovalently label and/or detect biological and nonbiological materials such as, for example, enzymes and other proteins, amino acids, antibodies, drugs, hormones, nucleotides and polysaccharides.
It is also an object of the invention to provide pH-insensitive fluorescent markers having high quantum yields and extinction coefficients, and therefore high fluorescence, compared with available fluorescent markers.
It is a further object of the present invention to provide fluorescent markers which are relatively photostable, which have sharp and distinct absorption and emission maxima, and which have relatively small Stokes shifts.
An additional object of the present invention is to provide a fluorescent compound having a general chemical structure which is easily modified by the addition or substitution of chemical moieties to, for example, change the reactivity of the compound, modify the compound's emissive and absorptive maxima and solubility in polar or nonpolar solvents, and covalently or noncovalently associate the compound with a material.